This antibiotic is described in U.S. Pat. No. 4,297,488, which illustrates different synthesis paths comprising carbamoylation of various desacetylated intermediates with various agents, using different protective groups on the carboxyl and/or on the amino group. It also describes the passage of enzymatic hydrolysis to produce certain of these derivatives, using acetil-esterase from citrus fruit peel, although with very slow reactions (6-15 hours) and without commenting on the quality of the products obtained. A similar process is also described by the same authors in Tetrahedron Lett 46, 4653-6 (1973), using protected intermediates such as p-nitrobenzyl esters, on which a deprotection and an enzymatic hydrolysis are carried out, to hence obtain 7-methoxy-3-desacetylcefalotin in carboxylic acid form. The product is then treated with chlorosulphonyl isocyanate to obtain cefoxitin.
The synthesis paths generally used on an industrial scale in modern chemistry produce optically pure cefoxitin, using 7-ACA (7-aminocephalosporanic acid) as raw material, and comprise four main steps, preferably carried out in the following order:
1. acylation of the amino group in position 7
2. introduction of the methoxy group in position 7α
3. removal of the acetyl group in position 3
4. carbamoylation of the hydroxyl group in 3 obtained in the preceding step.
Step 1 corresponds to the synthesis of cefalotin, a cephalosporin. This is then transformed into the corresponding cephalomycin by methoxylation to give the intermediate II (step 2):
and then desacetylated to give the compound I.
Although other sequences are possible, the aforedescribed is advantageous as it does not use protective groups either for the amino group or for the carboxyl group, and hence enables the number of necessary operations to be minimized, while enabling a raw material saving.
For example WO2004/083217A1 (page 6) describes the saponification of the intermediate II with sodium hydroxide in a water-methanol mixture cooling to −45° C.; this temperature is maintained for the entire duration of hydrolysis, and can be raised only on termination of the reaction, after neutralizing the base with acid.
The compound I is then isolated as the benzathine salt, after removing the methanol by distillation at moderate temperature; it is thus evident that 1) the use of methanol is justified by the need to reach very low temperatures for the reaction and 2) it is necessary to remove the solvent before isolating the product.
Alternatively the product can be extracted in solvent either in the undissociated form by acidification, or as a basic salt, for example tetrabutylammonium, as described in EP 1748049A2.
Cefoxitin can be obtained from the compound I by carbamoylation of the isiocyanate group, as described in the aforesaid patents, or with other isocyanates, as described for example in U.S. Pat. No. 4,292,427.
In all cases, given the lability of β-lactam structures and the need to operate under extremely basic conditions, the desacetylation of compound Ito give compound II is conducted at very low temperature to prevent product degradation; consequently organic solvents are used to lower the freezing point of the solutions.
These are therefore methods requiring the cooling of thousands of liters of solutions to a temperature of the order of −45° C., these temperatures to be maintained for the entire duration of the reaction, by using refrigeration machines or refrigerant fluids (such as liquid nitrogen); this results in a considerable energy cost. The solvent is then removed by distillation under vacuum by heating to +30/+40° C., with further considerable energy consumption (both for heating the solution and for the operation of the vacuum pump and condenser cooling).
It must also be considered that the use of solvents such as methanol involves danger due to solvent inflammability, possible operator intoxication, release of vapours into the environment, and drawbacks due to the production of methanol-containing aqueous effluents, which must be suitably disposed off.
A more ecocompatible path is therefore highly desirable, such as hydrolysis taking place only in water at ambient temperature. To effect such hydrolysis in a reasonable time and avoid product degradation a catalyst is required, for example an enzyme.
Enzymatic desacetylation of cephalosporins (not of cephamycins as in compound II) has been known for some time and has been described with enzymes of various origins.
For example acetyl esterase from wheat germ was described by Gilbert et al. in GB 1121308 (Glaxo, 1964), the enzyme present in citrus fruit peel was described by Jeffery et al in Biochem. J. 81, pages 591-6 (1961).
The aforesaid U.S. Pat. No. 4,297,488 describes the enzymatic hydrolysis of various cefoxitin synthesis intermediates, catalyzed by acetyl esterase from citrus fruit; however the method described therein is not applicable on an industrial scale, because of the poor performance of the catalyst. This is an enzyme of low specific activity involving very lengthy reaction times (6-15 hours described), difficult to produce as it derives from a poorly reproducible source subject to seasonal variations. Moreover it is applied in soluble form, is not recycled, and neither the purification nor the immobilization of the enzyme is described. Neither the yields of the acetyl derivatives obtained nor their quality are described.
Other enzymes active on cephalosporins have been discovered, starting from those involved in the biosynthesis path of cephalosporin C in Acremonium chrysogenum (or Cephalosporium acremonium) and in Streptomyces clavuligerus; these are considered as undesirable enzymatic activities, which lead to the formation of desacetyl cephalosporin C, a fermentation by-product. It should be noted that the biosynthesis of cephamycins in Nocardia lactamodurans and in Streptomyces clavuligerus does not comprise the hydrolysis of the acetyl group on a cephamycin (P. Liras, Antonie van Leeuwenhoek 75, 1999, pages 109-24); acetyl esterase activity on cephamycins is therefore not known, not even in cephamycin producer microorganisms.
Enzyme catalized hydrolyses of the acetyl group on 7-ACA or on cephalosporin C have been described, but not on cephamycins; in particular:
1) an esterase produced by Bacillus subtilis (Abbott and Fukuda, Antimicrob Agents Chemother 8, 3, pages 282-8, 1975 and Appl Microbiol, 30,3, pages 413-8 1975) is used in immobilized form for hydrolyzing 7-ACA to 3-desacetyl-7-ACA. The enzyme is sufficiently active and stable but tends to become detached from the immobilization support. Other authors (Takimoto et al. Appl Microbiol Technol 65, pages 263-7, 2004) describe the fermentation of this enzyme in recombinant Eschericia coli, its purification and immobilization on solid support and its use for producing 3-desacetyl-7-ACA.
2) The Rhodosporidium toruloides described by Politino et al. in Appl Environm Microbiol 63, 12, pages 4807-11, 1997, produces an enzyme active on 7-ACA, which can be used as catalyst in this reaction both in the form of a non-fermenting biomass (resting cells) and as an isolated and purified enzyme. The hydrolytic activity manifested by this enzyme on cefalotin is however low, only 34% of that on 7-ACA; Hydrolysis of cephamycins is not described. The same enzyme is also used by Chiang et al. (US 2002/0048781BMS, 2002) who describe a strain of recombinant Acremonium crysogenum, able to express acetyl esterase from Rhodosporidium, used to produce desacetylcephalosporin C directly in fermentation broths.
3) Another acetyl esterase is described by Venturi et al. in Appl Environ Microbiol 64, 2, pages 789-92, 1998: this is a xylan esterase produced by Bacillus pumilus, an enzyme connected with the degradation of xylans, which also shows activity on 7-ACA and on cephalosporin C; other publications describe expression of the same enzyme in coli. Activity on cephamycins is not described.
Hence enzymatic hydrolysis of the acetyl group of cephamycins has never been applied on an industrial scale in the state of the art; moreover, notwithstanding the wide literature available on a similar reaction in cephalosporins, an enzyme has not been described which is sufficiently active and stable for use on cephamycins.